Bifunctional chelators for sequestering lanthanides

ABSTRACT

The present invention relates to a method for preparing a bifunctional chelator for lanthanide. The method comprises the steps of providing a starting material which has an amino and carboxyl group; protecting the amino with an amino protecting group and the carboxyl with a carboxyl protecting group to produce a protected compound; reacting the protected compound with cyclen to generate a monoalkylated cyclen; reacting the monoalkylated cyclone with an activated compound to generated tetra-alkylated cyclone; removing the amino protecting group with a first protecting group removal reagent; and removing the carboxyl protecting groups with a second protecting group removal reagent to yield a bifunctional chelator having three more carboxyl groups and one or more amino groups.

TECHNICAL FIELD

The present invention relates to a method for synthesizing abifunctional chelator for sequestering lanthanides.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (“MRI”) for medical diagnostics can benefittremendously from use of contrast agents, which are gadoliniumcompounds. DOTMA and DOTA are well-studied molecules capable of bindinggadolinium ions (Gd³⁺), forming very stable complexes. Each complex canbind a water molecule and alter its relaxivity and hence be used as aMRI contrast agent. The DOTMA complex is superior.

However, neither DOTMA not DOTA are bifunctional, that is, neither canbe linked covalently to another molecule, meaning that they cannot beincorporated into polymers, attached to surfaces, or otherwise modifiedand targeted to a specific disease. There are a few bifunctionalderivatives of DOTA in the literature but they are very tedious to makeand even making 10 grams is difficult. The cost of buying such compoundscan be thousands of dollars per gram.

The present invention provides a first bifunctional DOTMA analog and arobust synthetic method for preparing this analog. The synthetic methodis scalable for readily producing 50-gram, 500 gram or more of thebifunctional DOTMA molecule. Moreover, this same chemistry is alsoapplicable to make a related bifunctional DOTA and DO3A analogs ascontrast agents with different properties.

SUMMARY OF THE INVENTION

In general, the bifunctional chelator of the present invention has achelating moiety with specific geometry and charge, and a side arm witha functional group, such as hydroxyl, thiol, amino, hydrazine, orcarbonyl, for attachment to a carrier, such as dendrimers,nanoparticles, and quantum dots. The chelating moiety may be anymolecules which have sufficient binding affinity for a lanthanideincluding DOTMA (1), DOTA (2), HP-DO3A (3), or analogs or derivativesthereof DOTMA (1) and DOTA (2) each have four carboxylates whereasHP-DO3A (3) has three carboxylates and one hydroxyl group.

The present invention provides a method for preparing a bifunctionalchelator for a lanthanide, which comprises the steps of providing astarting material which has an amino and carboxyl group; protecting theamino with an amino protecting group and the carboxyl with a carboxylprotecting group to produce a protected compound; reacting the protectedcompound with cyclen to generate a monoalkylated cyclen; reacting themonoalkylated cyclone with an activated compound to generatedtetra-alkylated cyclone; removing the amino protecting group with afirst protecting group removal reagent; and removing the carboxylprotecting groups with a second protecting group removal reagent toyield a bifunctional chelator having three more carboxyl groups and oneor more amino groups.

In one aspect, compounds 1 and 2 are designed to have four freecarboxylates and a highly nucleophilic primary aliphatic amino group forconjugation. The aliphatic amino group was expected to show greaternucleophilicity and attachment efficiency than does an aromatic aminogroup.²

In another aspect, amino acid esters are susceptible to base-catalyzedracemization. Because the products and intermediates containing thecyclen core are all bases, column chromatography on silica requiresrather polar solvents containing base to minimize streaking. In order toavoid epimerization (which was actually encountered in development ofthe successful route), column chromatography was to be avoided. Thissimplifies and speeds the synthesis, particularly on large scale.

In yet another aspect, the chelating moiety is the (RRRR)-stereoisomerof DOTMA.³ A diastereomeric mixture of products is a heterogeneouspopulation. Enantiomerically pure precursors and reactions whichpreserve e.e. are chosen carefully throughout.

In still another aspect, ester and primary amine protecting groups arechosen in the synthesis to be removed under different conditions. Thefour carboxylic acids are designed to be protected as methyl esters forsimpler NMR spectra and so that they are all removed with a singlereagent. The present invention provides

In further aspect, the organic synthetic method of the present inventionis used to construct bifunctional molecules having one end for bindinggadolinium tightly (a chelator) and other end being firmly andcovalently attached to a nanodevice, such as a dendrimeric nanodevice.Without these properly designed bifunctional molecules, the nanodevicecould fail for a number of reasons, including release of toxicgadolinium or lack of desired gadolinium-water interactions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows representative examples of bifunctional chelators forlanthanides.

FIG. 2 is a representative scheme for the synthesis of bifunctionalchelators for lanthanides.

FIG. 3 illustrates the attachment chemistry for bifunctional chelators.

FIG. 4 is a scheme for the synthesis of HP-DO3A.

DETAILED DESCRIPTION OF THE INVENTION

As used in this disclosure, the singular forms “a”, “an”, and “the” mayrefer to plural articles unless specifically stated otherwise.Furthermore, the use of grammatical equivalents of articles such as“functionalization”, “modification”, or “derivatization” is not meant toimply differences among these terms unless specifically indicated in thecontext.

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in analyticalchemistry, organic chemistry, material sciences, and nanotechnologydescribed herein are those well known and commonly employed in the art.Standard techniques, or modifications thereof, are used for chemicalsyntheses and chemical analyses of the present invention (see generally,March, “ADVANCED ORGANIC CHEMITRY: REACTIONS, MECHANISMS, ANDSTRUCTURE”, 3rd ed. (1985) John Willey & Sons, New York, N.Y.)

The synthesis of the bifunctional chelator of the present invention isshown in FIG. 2. To start the synthesis, commercially available(S)-4-amino-2-hydroxybutyric acid 4 was protected with a methyl groupfor the carboxyl group and a BOC group for the amino group, to give 5 asoil in about 90% yield. Notably, the protected compound 5 was purifiedreadily using silica gel chromatography. This reaction also produced aminor side-product (<5% yield) which has BOC groups on both the amineand hydroxyl groups. Other common protecting groups for both carboxyland amino groups may also be used as described bar Green and Wuts(Protective Groups in Organic Synthesis, 3^(rd), John Wiley & Sons, NewYork). For example, Cbz may be used in the place of Boc and tert-butylmay be used in the place of methyl.

The next step involves the activation of the hydroxyl group of compound5 for the alkylation of cyclen. In our early work, we found that thehydroxyl group of compounds such as 5 could be activated by conversionto a bromide (not shown), but that subsequent alkylation with cyclen wasvery slow, leading to partial racemization at the single chiral center.⁴Moreover, the methods available to make the bromide inverted theconfiguration at C-2, giving (R)-bromide and then the undesired (S)configuration of 7 after reaction with cyclen. Activation of thehydroxyl group as a mesylate solved the problem of double inversion, butthe alkylation step was just as slow as when using the bromide. Attemptsto activate the hydroxyl group as a triflate failed, because thetriflate was so reactive that the carbamate group in BOC- orCbz-protected 5 (R¹=BOC or Cbz) apparently attacked the triflate evenbefore cyclen could be added.

A satisfactory result was obtained for successful activation andcoupling with cyclen with only a single inversion by using a4-nitrobenzenesulfonate group, which was introduced within an hour usingDMAP catalyst.⁵ The solution of 6 so produced was then treated withexcess (3 equiv) of cyclen to assure a high degree of monoalkylation.After stirring overnight, the reaction mixture was washed with largeamounts of water to remove the excess cyclen. Compound 7 was isolated ina semi-crude form, suitable for the next step. The use of excess cyclen($35 per gram in 100 g quantities) may potentially be a drawback but itmay significantly simplify the purification process and increase thepurity of the product as well. Additionally, cyclen may also berecovered.

The triflate of commercially available (S)-lactic acid methyl ester wasmade in a way which avoids its purification by distillation or columnchromatography, giving almost quantitative yields without racemization.⁴Alkylation of semi-crude 7 with this triflate (3.3 equiv) in thepresence of K₂CO₃ was complete after overnight stirring. Not all KOTfcould be removed, however, even using aqueous washing of the solution of(RRRR)-8 in organic solvents. Thus, semi-purified (RRRR)-8 was convertedto a salt (RRRR)-10 using HCl in methanol, which kept the four methylester groups intact but removed the BOC group for future coupling to acarrier, such as a dendrimer. Analysis of both (RRRR)-8 and (RRRR)-10 by¹³C NMR on a 500 MHz spectrometer showed the presence of only a singleset of carboxylate carbons, consistent with both diasteromeric as wellas enantiomeric purity, since if any of the four chiral centersepimerized partially, more than a single set of resonances would havebeen seen. Similar treatment of compound 7 with methyl bromoacetate,followed by methanolic HCl, gave compound 11 of presumed(R)-configuration.

The method of the present invention provides several advantages. First,the method provides a high overall yield. Overall yields of (RRRR)-10and (R)-11 from 4 are all greater than 70%. The method is also simpleand scalable. The fact that all purifications after formation of 5 aredone by extraction or precipitation speeds synthesis and makeslarge-scale reactions feasible.

Both precursors (RRRR)-10 and (R)-11 have been successfully linkedthrough the free amino group to acylating agents. The tetraester saltsare also stable for storage, without danger of racemization because ofthe protonation state of the samples—they are essentially in a bufferedstate.

The synthesis of a fully deprotected compound (RRRR)-1 is shown in FIG.4. Treatment of either (RRRR)-8 or (RRRR)-10 with base in methanol-watermixture led to exclusive or extensive formation of lactam 13 at theexpense of (RRRR)-1, a totally unacceptable result since the side-chainamino group was now useless. However, treatment of (RRRR)-8 with aslight excess (4.5 equiv) of LiOH in THF-water led to clean hydrolysisof the methyl esters without any evidence for lactam formation.Subsequent deblocking of the side-chain Boc group proceeded withoutincident.

Lactam formation from 10 on basification could be a serious problem,because the free side-chain amino group would be unavailable as a linkerto a carrier, such as a nanodevice. However, basification of the salt of10 in the presence of acylating agent (e.g. DCC-activated carboxylicacid, carboxylic acid chloride, etc.) and in the absence of hydroxide(which could be the culprit in surprisingly facile removal of the BOCgroup during formation of 13) prevents lactam formation and ultimatelyleads to be attached to the desired nanodevices (schematicallyillustrated as 14 in FIG. 3)

The synthesis of a HP-DO3A analog 3 is illustrated in FIG. 4. Incompound 7, the single ester moiety is reduced to the alcohol 15 using areducing reagent, such as LiBH-₄, NaBH₄, or LiAlH₄. LiBH₄ and NaBH₄ havebeen shown to reduce BOC- or Cbz-amino acid methyl esters withoutreduction of the carbamate.⁶ LiAlH₄, under carefully controlledconditions, has been shown to function similarly.⁷ Subsequentconversions of compound 15 follows those done in FIG. 2.

In anther aspect of the present invention, the analysis of intermediatesand products for potentially interfering impurities is of primeimportance. For example, it is desired that final products arecompletely free of any other primary amine besides the one desired,because any mixture of amines may lead to heterogeneous mixtures ofcoupling products. This may lead to complications such as a dendrimersurface with some sites unavailable to gadolinium binding, orheterogeneous populations of dendrimers with variable chemical andphysical properties. The impurities may be identified by using NMR, suchas 500 MHz NMR, HPLC, or LC-MS. For example, HPLC or LC-MS (Finnigan LCQelectrospray-MS) was used to detect the presence (if any) of unreactedcompound 5 in semi-crude 7 or subsequent intermediates.

Potentially interfering impurities may also be synthesized if necessaryand conclusively identified. For example, DMAP catalyst is present insemi-crude 7. Its fate in the subsequent step—exposure to strongalkylating agents such as the triflate or bromide—is unknown—thoughalkylation of pyridines in general is a common reaction. Therefore, inseparate experiments, DMAP is alkylated with these agents, and theresulting products are identified using standard techniques. Finally,samples of (RRRR)-8 and (R)-10 is analyzed for these potentialside-products and it needled additional purification steps may beincluded in their synthesis.

All compounds need to be fully characterized, including NMR, MS, HPLC,elemental analysis, and optical purity determination (e.e), such asusing chiral HPLC or optical rotation.

Though the tetraester salts (RRRR)-10 and (R)-11 appear to be chemicallystable for months in tightly sealed containers in desiccators, thematerials were produced as extremely hygroscopic powders. Even in therelatively dry air of San Diego, the powdery material would start tobecome sticky within minutes of exposure to laboratory air. Absorptionof water may cause chemical changes, for example hydrolysis: in fact, wehave monitored samples of 10 and 11 in CD₃OD-D₂O solutions and findafter one day at 25° C. evidence for release of CH₃OD from methyl esterhydrolysis. Short exposure to lab air would not be expected to lead tosignificant hydrolysis, but if the samples pick up water their masschanges and with it the percent composition by weight of desiredchelator, which if ignored would lead to errors in stoiChiometry duringattempted nanodevice construction.

To avoid all these problems, the bifunctional chelator of the presentinvention may be formulated into various salt forms, including, but notlimited to, bromide or chloride. This can be simply achieved using HBror HCl in methanol during the removal of the BOC group. Alternatively,the bifunctional chelator may also be packaged in standard sizes foruse.

The examples set forth above are provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the compositions, and are notintended to limit the scope of what the inventors regard as theirinvention. Modifications of the above-described modes for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

The invention also provides the following compositions:

In alternative embodiments, a compound of the invention comprises aformula as set forth in FIG. 1, or a compound made by the synthesismethod of FIG. 2 or FIG. 4, or a compound of FIG. 3.

In alternative embodiments, a compound of the invention, e.g., abifunctional chelator of the invention, has (comprises) a chelatingmoiety with specific geometry and charge, and a side arm with afunctional group, such as hydroxyl (A), thiol (B), amino (C), hydrazine(D), carbonyl (E), alkene (F), alkyne (G) or azide (H), for attachmentto a carrier, such as dendrimers, nanoparticles, or quantum dots. Suchcompositions of matter comprise the structures illustrated and describedherein (e.g., as shown above).

The invention provides carrier, dendrimers, nanoparticles and/or quantumdots comprising the structures illustrated and described herein.

In alternative embodiments of a compound of the invention, e.g., animaging agent of the invention, the part of the side-chain shown as(CH₂)_(n) may be from one to ten atoms long (n=1 to 10) and that any ofthe carbons may be further independently substituted. In alternativeembodiments, any of the H on the Z group may be independentlysubstituted by alkyl or aryl groups.

The compositions can be used as contrast agents in any interventionaltherapy; e.g., including the targeting of an undesired tissue or tissuecomponent with high thermal energy using focused ultrasound (e.g., Clineet al., “MR Temperature Mapping of Focused Ultrasound Surgery,” Mag.Resn. Med., 31:628 6136 (1994)), radiofrequency generators (e.g., Rossiet al., “Percutaneous RF Interstitial Thermal Ablation in the Treatmentof Hepatic Cancer,” AJR, 167:759 768 (1996)), microwave antennae (e.g.,Schwarzmaier et al., “Magnetic Resonance Imaging of Microwave InducedTissue Heating,” Mag. Resn. Med., 33:729 731 (1995)), and lasers (e.g.,Vogl et al., “Recurrent Nasopharyngeal Tumors: Preliminary ClinicalResults with Interventional MR Imaging-Controlled Laser-InducedThermotherapy,” Radiology, 196:725 733(1995)); the use of cryoablation(i.e., liquid nitrogen) and the injection of denaturing liquids (e.g.,ethanol, hot saline) directly into the undesired tissue (e.g., Nagel etal., “Contrast-Enhanced MR Imaging of Hepatic Lesions Treated withPercutaneous Ethanol Ablation Therapy,” Radiology, 189:265 270 (1993)and Honda et al., “Percutaneous Hot Saline Injection Therapy for HepaticTumors: An Alternative to Percutaneous Ethanol Injection Therapy,”Radiology, 190:53 57 (1994)); the injection of chemotherapeutic and/orchaotropic agents into the tissue (e.g., Pauser et al., “Evaluation ofEfficient Chemoembolization Mixtures by Magnetic Resonance Imaging ofTherapy Monitoring: An Experimental Study on the VX2 Tumor in the RabbitLiver,” Cancer Res., 56:1863 67 (1996)); and photodynamic therapies,wherein a cytotoxic agent is activated in vivo by irradiation with light(e.g., Dodd et al., “MRI Monitoring of the Effects of PhotodynamicTherapy on Prostate Tumors,” Proc. Soc'v Mag. Resn., 3:1368, ISSN 10659889 (Aug. 19 25, 1995)). The shared goal of all such interventionaltherapies is the treatment of undesirable tissue or tissue component(i.e., cancerous, tumor, neoplastic tissue or tissue component) bycausing the necrosis, ablation, coagulation, or denaturation of suchtissue; or, e.g., as described in U.S. Pat. No. 7,175,829.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and this description. Other features, objects,and advantages of the invention will be apparent from the descriptionand drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

REFERENCES

-   1. Renn, O. and Meares, C. F. “Large-Scale Synthesis of the    Bifunctional Chelating Agent    2-(p-Nitrobenzyl)-1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic    Acid, and the Determination of Its Enantiomeric Purity by Chiral    Chromatography,” Bioconjugate Chem. (1992) 3, 563-569.-   2. Chappell, L. L., Rogers, B. E., Khazaeli, M. B., Mayo, M. S.,    Buchsbaum, D. J. and Brechbiel, M. W. “Improved Synthesis of the    Bifunctional Chelating Agent    1,4,7,10-Tetraaza-N-(1-carboxy-3-(4-nitrophenyl)propyl)-N′,N″,N′″-tris(acetic    acid)cyclododecane (PA-DOTA),” Bioorg. Med. Chem. (1999) 7,    2313-2320.-   3. Woods, M., Aime, S., Botta, M., Howard, J. A. K., Moloney, J. M.,    Navet, M., Parker, D., Port, M. and Rousseaux, O. “Correlation of    Water Exchange Rate with Isomeric Composition in Diastereoisomeric    Gadolinium Complexes of Tetra(carboxyethyl)dota and Related    Macrocyclic Ligands,” J. Am. Chem. Soc. (2000) 122, 9781-9792.-   4. Effenberger, F., Burkard, U. and Willfahrt, J. “Enantioselektive    Synthese N-substituierter □-Aminocarbonsäuren aus    □-Hydroxycarbonsäuren,” Liebigs Ann. Chem. (1986) 314-333,-   5. Hoffman, R. V. and Kim, H.-O. “Preparation of (R)-2-Azidoesters    from 2-((p-Nitrobenzene)sulfonyl)oxy Esters and Their Use as    Protected Amino Acid Equivalents for the Synthesis of Di- and    Tripeptides Containing D-Amino Acid Constituents,”    Tetrahedron (1992) 48, 3007-3020.-   6. Salituro, F. G., Agarwal, N., Hofmann T. and Rich, D. H.    “Inhibition of aspartic proteinases by peptides containing lysine    and ornithine side-chain analogs of statine,” J. Med. Chem. (1987)    30, 286-295.-   7. Namba, K., Shinada, T., Teramoto, T. and Ohfune, Y. “Total    Synthesis and Absolute Structure of Manzacidin A and C,” J. Am.    Chem. Soc. (2000) 122, 10708-10709.

1. A method for preparing a bifunctional chelator for a lanthanide,comprising: (a) providing a starting material which has an amino andcarboxyl group; protecting the amino group with an amino protectinggroup and the carboxyl with a carboxyl protecting group to produce aprotected compound; reacting the protected compound with cyclen togenerate a monoalkylated cyclen; reacting the monoalkylated cyclone withan activated compound to generated tetra-alkylated cyclone; removing theamino protecting group with a first protecting group removal reagent;and removing the carboxyl protecting groups with a second protectinggroup removal reagent to yield a bifunctional chelator having three ormore carboxyl groups and one or more amino groups.
 2. A compoundcomprising (a) a formula selected from the group consisting of one ofthe following formulas:

a compound as set forth in FIG. 1, a compound made by the synthesismethod of FIG. 2 or FIG. 4, and a compound of FIG. 3; (b) the compoundof (a), wherein the (CH₂)_(n) comprises from one to ten atoms long (n=1to 10), (c) the compound of (a) or (b), wherein any of the carbons isfurther independently substituted, (d) the compound of (a), (b) or (c),wherein any of the H on the Z group is substituted with an R₁ groupindependently selected from the group consisting of hydrogen, halo,hydroxy (—OH), thiol (—SH), cyano (—CN), formyl (—CHO), alkyl, aryl,haloalkyl, alkenyl, alkynyl, amino, nitro (—NO₂), alkoxy, haloalkoxy,thioalkoxy, alkanoyl, haloalkanoyl and carbonylox, or (e) the compoundof any of (a) to (d), wherein the R group is substituted with an R₁group independently selected from the group consisting of hydrogen,halo, hydroxy (—OH), thiol (—SH), cyano (—CN), formyl (—CHO), alkyl,aryl, haloalkyl, alkenyl, alkynyl, amino, nitro (—NO₂), alkoxy,haloalkoxy, thioalkoxy, alkanoyl, haloalkanoyl and carbonyloxy.
 3. Abifunctional chelator comprising a compound as set forth in claim
 2. 4.The compound of claim 2: (a) further comprising a chelating moiety, (b)the compound of (a), wherein the chelating moiety has a specificgeometry and charge, (c) the compound of (a) or (b), wherein and thecompound comprises a side arm with a functional group, (d) the compoundof (a), (b) or (c), wherein the side arm comprises a hydroxyl (A), thiol(B), amino (C), hydrazine (D), carbonyl (E), alkene (F), alkyne (G) orazide (H), (e) the compound of any of (a) to (d), wherein the side armis used for attachment to a carrier, or (f) the compound of any of (a)to (e), wherein the side arm is used for attachment to a dendrimer, ananoparticle, or a quantum dot.
 5. A dendrimer, a nanoparticle, or aquantum dot a compound comprising a compound as set forth in claim
 2. 6.A magnetic resonance imaging (MRI)-enhancing compound (contrast agent)comprising a dendrimer, a nanoparticle, or a quantum dot of claim 5, acompound as set forth in claim 2, or a bifunctional chelator of claim 3.7. A method for enhancing the image of a magnetic resonance imaging(MRI) comprising administration to an individual the magnetic resonanceimaging (MRI)-enhancing compound (contrast agent) of claim
 6. 8. Amethod for monitoring treatment of a tissue or for evaluating efficacyof an interventional therapy in an individual, said method comprising:(i) a) administering the magnetic resonance imaging (MRI)-enhancingcompound (contrast agent) of claim 6, wherein; b) subjecting theindividual to magnetic resonance imaging (MRI) to determine an initialsignal intensity value in a region of interest of said undesired tissue;c) applying an interventional therapy to at least a portion of saidundesired tissue in order to treat said undesired tissue; and d)monitoring with magnetic resonance imaging a change in said initialsignal intensity value in said region of interest of said undesiredtissue; (ii) the method of (i), wherein the tissue is cancerous tissue,tumor tissue or neoplastic tissue, (iii) the method of (i) or (ii),wherein the interventional therapy is a thermal energy generation, acryoablation, an injection of a denaturing liquid, an injection of achemotherapeutic agent or a photodynamic therapy; or (iv) the method ofany of (i) of (iv), wherein the interventional therapy is the generationof thermal energy, wherein the thermal energy is generated by a sourcecomprising a focused ultrasound wave, a radiofrequency wave, a microwaveand/or a laser.
 9. The method of claim 8, further comprising: e)stopping the interventional therapy application when a change in theinitial signal intensity value in the region of interest of theundesired tissue is more than about a 10% reduction in the initialsignal intensity value.